The long term goal of this research is the study of regulation of pathways of folate metabolism in both normal and diseased states. An approach to the study of metabolic regulation is the characterization of proteins or protein complexes which catalyze reactions at branch points in metabolism, and which thus regulate the flux through competing pathways. We propose to study methylenetetrahydrofolate reductase, a flavoprotein which regulates a key branch point in folate metabolism. This enzyme catalyzes the first step in the flow of one carbon units from methylenetetrahydrofolate into the pathway for de novo biosynthesis of methyl groups. A procedure is outlined for purification of this enzyme, and for the study of its catalytic mechanism using steady-state and rapid reaction kinetic measurements and anaerobic titrations. The regulation of reductase activity by S-adenosylmethionine will be examined by kinetic studies, and the effects of other metabolites in folate-dependent pathways on reductase activity will be examined. Preliminary results indicate that this enzyme co-purifies with methionine synthetase, a B12 containing enzyme which catalyzes the next reaction in the pathway for biogenesis of methyl groups. Experiments are described for establishing whether methionine synthetase forms a complex with methylenetetrahydrofolate reductase, and if so, for the characterization of the complex and its component proteins. The effect of complex formation on the physical and catalytic properties of the reductase, and on its regulation will be studied. Using electron spin resonance probes in combination with the rapid freeze apparatus and rapid reaction kinetic studies, the reaction mechanism of the B12-dependent methionine synthetase will be studied, and the role of cobalt in catalysis will be examined. These studies may have implications for our understanding of the complex inter-relationships between folate and B12 metabolism seen in pernicious anemia.